GSM7839405: iPSC-derived HOs cultured in three conditions; Homo sapiens; RNA-Seq
The reagents were prewarmed at 37ºC. Six organoids (D-45) were recollected in a 15 mL polypropylene (Falcon) tube to wash with PBS twice or thrice. As a dissociation reagent, 1 mL of trypsin 0.5% - EDTA (10X) (Gibco) with 50 µM Y-27632 (Gibco), 40 U/µL RNase inhibitor (Invitrogen), and 1 U DNase I (Thermo Scientific) was added to the organoids and kept on 37ºC in a Vari-Mix™ Platform Rocker (Thermo Fisher) at 30 rpm for 15 min. Every 5 min, we mechanically disrupted the organoids till it was homogenized into single cells. Afterward, we proceeded to the inactivation of trypsin with 1 mL Advanced DMEM/F12 mix containing Bovine Albumin Fraction V (7.5% solution, Gibco) 50 µM Y-27632 (Gibco), 40 U/µL RNase inhibitor (Invitrogen), and 1 U DNase I (Thermo Scientific). We collected and filtered the 2 mL through a 40 µm Cell Strainer (Corning) into a 50 mL tube (Falcon). We then add another 1 mL Advanced DMEM/F12 mix to recover any left cells from the strainer. We carried out the fixation of at least 10^5 required single cells, following the protocol described in the EvercodeTM fixation kit (Parse Bioscience). Library construction was performed under manufacturer's protocol (Parse Bioscience)